PROJECT SUMMARY Cell surface and secreted glycoproteins and glycolipids play critical roles in cellular physiology and pathology, but challenges remain in understanding how glycan-based reagents can be leveraged for biochemical and therapeutic purposes. A recent National Academies study concluded that there are numerous critical unmet needs for transformative methods and tools to fill technology gaps for carbohydrate-based applications in health, energy and materials science. One of the most critical remaining hurdles has been accessibility to mammalian enzymes required for glycan synthesis, modification, and catabolism (glycoenzymes). These glycoenzymes are essential for synthesis and modification of glycan- and glycoprotein-related therapeutics and biologics in the pharmaceutical industry, development of applications in biological and biomedical glycoscience, production of analytical standards, and advancing our understanding of fundamental biochemical processes. Access to this diverse set of enzymes will solve an unmet need for these tools in a global glycomics/glycobiology market that is predicted to reach $2.1 billion by 2025. In prior studies, the Moremen lab at the University of Georgia developed strategies for recombinant production of all known human glycoenzymes (~350 target enzymes) by expression in mammalian cells. The broad utility of these enzymes was demonstrated for numerous biomedical applications and NIH program staff strongly suggested the formation of a commercial entity for the production of these enzymatic reagents. A biotech start-up company, Glyco Expression Technologies, Inc., was launched in 2018 to enhance the production, validation, and dissemination of these key reagents based on the proof-of-concept studies for mammalian expression developed at the University of Georgia. Thus, we propose Phase 1 Specific Aims that include: (Aim 1) enhanced production of challenging glycoenzymes using custom cell lines, new vector designs, and co-transfection strategies, (Aim 2) maximized recombinant human glycoenzyme expression, purification, and processing, and (Aim 3) optimized and validated enzyme activity, specificity, and storage of recombinant enzymes. These aims will advance the goals of developing critical tools to overcome major roadblocks in mammalian glycobiology and fulfill critical unmet needs to provide technologies essential for carbohydrate-based applications. These transformative reagents will be delivered in stable and well- characterized formats with appropriate documentation for enzyme specificity and will provide a framework for the developing biological and biomedical applications that expand our knowledge in glycochemistry and glycobiology and advance carbohydrate-based therapeutics.